A human artificial chromosome (HAC) vector was constructed from a 1 megabase yeast artificial chromosome (YAC) that was isolated by screening a randomly chosen subset of the CEPH YAC library for alpha satellite DNA. This YAC was modified to contain human telomeric DNA and a putative origin of replication from the human beta-globin locus. The resultant HAC vector was introduced into human cells by lipid-mediated DNA transfection, and HACs were identified which were mitotically stable in the absence of selection for at least 100 generations. Microdissected HACs used as FISH probes localized to the HAC itself and not to the arms of any endogenous human chromosomes, suggesting that the HAC was not formed by telomere fragmentation. After transfer to mouse cells, the HACs were mitotically unstable in the absence of selection, suggesting that there may be important functional differences between human and mouse chromosomes. In addition to the formataion of HACs, one fate of the HAC DNA was integration into the host cell genome leading us to question the functionality of the telomeres. In an effort to improve the rate at which the HAC DNA seeds functional telomeres, we are assessing whether the yeast telomeric sequences which are added to the HAC during propagation, adversely affect the efficieny of HAC formation. We have propagated our HAC vector in a yeast strain with a modified telomerase template which can add human telomeric DNA to the ends of the vector and will assess the rate of HAC formation. In addition, the HACs were clearly larger than the input vector DNA and their heterogenous composition made characterization of their content difficult. In an effort to increase the likelihood that functional HACs would form without host cell modification of the original HAC vector DNA. we have undertaken a new strategy using mapping information from the pericentromeric region of chromosome 16. A series of plasmid vectors have been constructed that can be used to isolate the chromosome 16 centromere directly from a monochromosomal hybrid ceell line by transformation-associated recombination (TAR) cloning. A GFP cassette has been included on one plasmid arm that should allow for flow sorting of tranfected cells. In addition, selectable markers are included on both arms for stable clone isolation. These plasmids are being used to isolate large contiguous tracts of alpha satellite DNA from chromosome 16, which will then be tested for their ability to form functional HACs in human cells. Our ability to manipulate these HAC vectors by recombinant genetic methods should allow us to further define the elements necessary for mammalian chromosome function. - genetics gene therapy human genome research